Alzahrani Alhusain J, Vallely Pamela J, McMahon Raymond F T
Division of Virology, Laboratory Medicine Academic Group, School of Medicine, University of Manchester, Clinical Sciences Building, Manchester Royal Infirmary, Oxford Road, M13 9WL, Manchester, UK.
J Virol Methods. 2002 Jan;99(1-2):53-61. doi: 10.1016/s0166-0934(01)00383-4.
Hepatitis C virus (HCV) is a major cause of chronic hepatitis with liver-related death occurring in 20-25% of patients who develop cirrhosis. Detection of the virus RNA in liver is difficult since viral expression is very low. In situ polymerase chain reaction (PCR) in situ hybridisation (ISH) was developed for detection and localisation of viral RNA in formalin-fixed, paraffin-embedded liver tissue. Nested PCR technology was adapted for in situ use employing primers derived from the 5' end of the HCV genome. Seventeen liver blocks from known HCV-infected patients were examined. Viral RNA was detected in liver from eleven patients using solution phase reverse transcriptase-PCR. Of these positive samples, ten were positive by the in situ method. Positive signal was detected in the cytoplasm of hepatocytes and mononuclear cells. No Kupffer cell or bile duct epithelial positivity was observed. No positive signal was evident in any of the negative controls. A reliable method is described to demonstrate the presence and localisation of HCV RNA in liver tissue using an in situ PCR-ISH assay and it is believed that this will be a valuable tool for the understanding of HCV replication and pathogenesis.
丙型肝炎病毒（HCV）是导致慢性肝炎的主要原因，在发展为肝硬化的患者中，20%-25%会出现与肝脏相关的死亡。由于病毒表达水平很低，在肝脏中检测病毒RNA很困难。原位聚合酶链反应（PCR）原位杂交（ISH）技术被开发用于在福尔马林固定、石蜡包埋的肝组织中检测和定位病毒RNA。采用源自HCV基因组5'端的引物，将巢式PCR技术应用于原位检测。对17个来自已知HCV感染患者的肝组织块进行了检查。使用液相逆转录-PCR在11名患者的肝脏中检测到病毒RNA。在这些阳性样本中，有10个通过原位方法检测为阳性。在肝细胞和单核细胞的细胞质中检测到阳性信号。未观察到库普弗细胞或胆管上皮细胞呈阳性。在任何阴性对照中均未发现明显的阳性信号。本文描述了一种使用原位PCR-ISH检测法来证明肝组织中HCV RNA存在及定位的可靠方法，并且认为这将是理解HCV复制和发病机制的一个有价值的工具。
